Advances in cell culture and recombinant DNA technologies have facilitated the expression of a variety of proteins of therapeutic or other economic value using genetically engineered cells. The expression of many biologically active therapeutic proteins, which are derived from higher eukaryotic sources, often requires specific post-translational modifications which do not naturally occur in lower eukaryotic or prokaryotic cells, thus necessitating the use of cells derived from higher eukaryotic sources. For example, the expression of glycoproteins in mammalian cells has the advantage of providing proteins which contain natural glycosylation. Mammalian-produced glycoproteins contain outer chain carbohydrate moieties, which are markedly different from the outer chain carbohydrate moieties present on glycoproteins produced from lower eukaryotes. The use of mammalian cells as hosts for the production of secreted mammalian proteins has the significant advantage over secretion from lower eukaryotes in that mammalian cells have a secretory system that readily recognizes and properly processes secretion-directed proteins, which is not necessarily true for lower eukaryotes.
Scaffold Attachment Regions (SAR), also called Matrix Attachment Regions (MAR) or Scaffold/Matrix Attachment Regions (S/MAR) are non-consensus-like AT-rich DNA elements several hundred base pairs (bp) in length, which organize the nuclear DNA of the eukaryotic genome into some 60,000 chromatin domains, 4-200 kbp loops, by periodic attachment to the protein scaffold or matrix of the cell nucleus. S/MARs have been isolated from regions surrounding actively transcribed genes but also from introns, centromeres and teleomeric regions and have been found to collaborate with enhancers to help regulate transcription by controlling the chromatin state of DNA. The observations that S/MARs positively interact with enhancers, form loop domains and often are located at the borders of transcriptionally active domains have led to the idea of using S/MARs as flanking elements around transgenes, forming so called mini-domains, in order to protect transgenes or expression cassettes from transcriptional silencing and the effects of surrounding heterochromatin (transcriptionally inactive chromatin) as well as possibly increase gene expression. Several publications have shown that S/MARs in a flanking position can strongly stimulate expression of transgenes, as well as reduce expression variability between cell clones (position effects). Moreover, due to the character of a mini-domain, expression should be independent from the integration site.
U.S. Pat. No. 5,985,607 discloses the use of certain SAR elements for expression of EPO and tPA. In Biochemistry, 30:1264-1270, 1991, the effect of other SAR elements on expression of the human interferon β is disclosed. Phi-Van et al. in Mol. Cell. Biol, 10:2302-2307, 1990 (“The chicken lysozyme 5′ matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes”) discloses the influence on gene expression of a MAR element located upstream of the chicken lysozyme gene. WO/9704122 discloses a method for producing polypeptides in plants with possible use of nuclear scaffold attachment region sequences and WO 02/14525 discloses an animal expression vector comprising β-globulin MAR or SAR sequences.
The proteins involved in the clotting cascade, including, e.g., Factor VII, Factor VIII, Factor IX, Factor X, and Protein C, are proving to be useful therapeutic agents to treat a variety of pathological conditions. Because of the many disadvantages of using human plasma as a source of pharmaceutical products, it is preferred to produce these proteins in recombinant systems. The clotting proteins, however, are subject to a variety of co- and post-translational modifications, including, e.g., asparagine-linked (N-linked) glycosylation; O-linked glycosylation; and γ-carboxylation of Glu residues. For this reason, it is preferable to produce them in mammalian cells, which are able to modify the recombinant proteins appropriately. Production of recombinant proteins within mammalian cells can be difficult because of a low genetic stability of the recombinant gene and/or silencing of the recombinant gene. Several molecular mechanisms have been reported that may lead to gene silencing, e.g., DNA methylation and histone deacetylation.
Thus, there is a need in the art to overcome the deficiencies of the known methods for making clotting proteins by providing mammalian production strains with a higher genetic stability in large-scale production to produce industrial quantities of the clotting proteins, particularly recombinant human Factor VII or Factor VII-related polypeptides.